Insulin-like signaling pathway: flies and mammals

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Insulin-like signaling pathway flies and mammals

• AS300-002 Jim Lund

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Insulin-like signaling responds to environmental
signals
Environmental signals Food, dauer hormone
DAF-2 receptor
INS-7
insulin-like hormone
sensory system
This feedback loop may allow all the cells to
make the same developmental or lifespan decision
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Drosophila insulin-like signaling pathway (ISP)

• Genes in this pathway were first investigated for
  effects on growth and size.
• ISP also affects blood sugar levels in the fly.
• Fly has five insulin-like proteins.
• Expressed strongly in small clusters of cells
  (IPCs) in the brain.
• Ablation of IPCs causes retarded growth and
  higher carbohydrate levels (Rulifson et al.,
  2002).

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Drosophila insulin-like signaling pathway (ISP)

• The gene InR is an insulin-like receptor in fruit
  flies.
• It is homologous to insulin receptors in mammals
  and to daf-2 in worms.
• Studied InR gene variants (alleles) in flies.
• (Tatar et al., 2001)

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InR various allele combinations produce
different results

• Some had a reduced survival rate
• Females in one type extended life span by 85
• Males followed the female pattern in most cases
• Not all the InR alleles extend longevity because
  the gene is highly variable.
• Some alleles produced developmental defects that
  carry over into adults.

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InR various allele combinations produce
different results
InREC34/InRE19, InRGC25/InRE19, InRE19/InRE19,
and /InRp554 Females A, B Males C, D
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Fly insulin-like signaling pathway

• Fly homolog of daf-16 dFOXO.
• Forkhead box DNA binding domain amino acid
  identity is between 74 and 86 percent.
• Akt phosphorylation sites are also well conserved
• dFOXO heterozygotes supress InR lifespan
  extension.

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Fly insulin-like signaling pathway

• Fly has four homologs of the PI3K age-1.
• Each controls different cellular processes.
• Increased signaling complexity in the fly
  relative to the worm.
• More complicated in human, 16 PIK3 genes.

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Fly and worm insulin-like signaling pathways
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Drosophila ISP regulation of lifespan
Nature 429 562-66, 2004
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Mammal insulin-like signaling pathway

• In vertebrates, the insulin receptor regulates
  glucose metabolism, while IGF-1R promotes growth.
  
• IGF-1R is activated by its ligand IGF-1, which is
  secreted in response to growth hormone.
• Pathway more complicated more tissue specific
  signaling and regulation.
• Multiple homologs, some specific to certain
  somatic tissues.
• Genetic investigation is more complicated.

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Mammal insulin-like signaling pathway

• In mice, inactivation of the growth hormone
  receptor decreases circulating IGF-1, impairs
  growth development, and increases lifespan.
• Calorie restriction, the only intervention
  demonstrated to reliably and consistently
  increase mammalian lifespan, always reduces
  circulating IGF-1.

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Mammal gene knock-out technology

• Recall that most organisms have two copies of
  each gene, one inherited from each parent.
• Using genetic engineering methods, it is possible
  to delete or otherwise alter one or both copies
  of a gene, so that the animal has either one or
  no working copy of the gene.
• A mouse altered in this way is called a
  "knock-out" mouse.

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Mammal gene knock-out technology
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Mammal insulin-like signaling pathway

• When both copies are knocked out, it is called a
  homozygous null mutant, or a double knock-out.
• An IGF-1R double knock-out is annotated Igf1r -/-
• When one copy of IGF-1R is knocked out, it is
  called a single knock-out, annotated Igf1r /-.
• Horzenberger created Igf1r -/- and Igf1r /-
  mice. The double knock-out Igf1r -/- mice did not
  survive. The single knock-out Igf1r /- mice
  survived.

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Igf1 knock-out mice

• The single knock-out Igf1r/- mice lived an
  average of 26 longer than wild-type mice.
• Female Igf1r/- mice lived an average of 33
  longer than wild-type,
• Male Igf1r/- mice lived an average of 16
  longer.

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ISP in Rats

• Rats with reduced GH/IGF-1 levels.
• 40 reduction in IGF-1 levels produces a 9-13
  lifespan extension.
• (Shimokawa et al., 2003)

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Mammal ISP cellular processes controlled
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Centenarian genetics human INSR

• INSR
• Study of 122 Japanese semisupercentenarians
  (older than 105) with 122 healthy younger
  controls.
• One INSR haplotype, which was comprised of 2 SNPs
  in linkage disequilibrium, was more frequent in
  semisupercentenarians than in younger controls.
• Kojima et al., 2004

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Insulin/IGF-1 Signaling Pathway Human Homologies
With Nematode, Flies And Mice
MOUSE
NEMATODE
FLY
Insulin/IGF-1
Ligand
Ligand
GH
INS/IGF1 R
INR b
INR
DAF-2
GHR (-/-)
Chico
IRS1-2
?
p85/p110a-b
Dp110/p60
AGE 1
?
Forkhead transcription factor
DAF-16
GLUCOSE METABOLISM
DEVELOPMENT
LONGEVITY
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Mammalian ISP

• Mouse mutants with reduced insulin signaling live
  longer.
• Mouse IGF-1 receptor mutant heterozygotes (ie.
  reduced IGF-1 receptors).
• Dog breeds with low levels of IGF-1 live longer
• Caloric restriction reduces insulin and IGF-1
  (increases mammal longevity)

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Mammalian Models

• Long-lived mice (like the worms) have been
  characterized by a deficiency in growth hormone
  and IGF-1.
• Tissue-specific inactivation of the insulin
  receptor has been experimentally effective
• Fat cells in mice 20 increase.
• Partial receptor inactivation also effective in
  mice .
• Deletion of the p66shc protein in mice results in
  a 30 increase in longevitythese mice can better
  withstand oxidative stress.
• Also, cells from these animals have lower levels
  of oxidants.
• P66shc appears to regulate the mammalian
  Forkhead-family member counterpart of DAF-16.

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Human ISP

• 7 human homologs of daf-16, the Forkhead family
  transcription factor.
• Called FOXOs or FKHRs.
• Several FOXOs are tumor suppressors, as are AKT
  and PTEN.
• Activation of FOXOs lead to
• cell cycle arrest, stress resistance, or
  apoptosis.

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Human ISP
Greer and Brunet, 2005
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Functions of human FOXOs
Greer and Brunet, 2005
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Insulin-like signaling pathway (ISP)